The intestinal epithelium is continuously exposed to high concentrations of bacteria. Intestinal epithelial cells (enterocytes) express pathogen pattern recognition Toll-like receptors (TLR), and produce inflammatory factors in response to stimulation with luminal bacteria or their TLR ligands. These responses are critical for gut maintenance because they promote mutualistic relationship between epithelium and commensal flora. Although intestinal epithelium can sense bacteria, it normally avoids dramatic inflammation by developing tolerance, a long lasting inhibition of inflammatory responses. Failure to develop or maintain tolerance to commensal bacteria can be regarded as an underlying cause of the intestinal inflammatory disorders. Unfortunately, little is known about the mechanisms of epithelial tolerance to bacteria. Our long-term goal is to understand the mechanisms of intestinal tolerance to commensal bacteria. We have found that bacterial lipopolysaccharide (LPS)-induced expression of the A20 deubiquitinase is necessary and sufficient for the development of tolerance to LPS, the TLR4 ligand, in enterocytes. A20 induction may be also responsible for enterocyte tolerance and cross-tolerance to the ligands of TLR3 and TLR9, but not TLR5 ligand flagellin (Fla). Because Fla induces the CYLD deubiquitinase, but not A20, it is possible that CYLD specifically inhibits TLR5 signaling. We hypothesize that A20 and CYLD promote tolerance to ligands of TLR3/TLR4/TLR9 and TLR5, respectively, which will be tested in 2 specific aims: 1. Assess role of A20 in tolerance to TLR ligands in intestinal epithelium by determining responses to different TLR ligands in enterocytes following forced expression or small interfering RNA (siRNA) silencing of A20. 2. Determine the role of CYLD in enterocyte tolerance to Fla. Induction of CYLD mRNA and protein by various TLR ligands will be measured using Northern and Western blots. Effects of CYLD ectopic expression and siRNA silencing on activation of inflammatory signaling by different TLR ligands will be used to elucidate the role and specificity of inhibitory effect of this deubiquitinase. CYLD expression in normal and inflamed intestine will be examined using immunofluorescence microscopy. This pilot study will define the key processes responsible for the development of tolerance to different TLR ligands in the intestine. Dissecting the mechanisms of intestinal tolerance will lead to better understanding of the pathogenesis of the intestinal inflammatory disorders such as necrotizing enterocolitis, and, ultimately, better therapies. Intestinal inflammatory disorders are caused by failure to establish and/or maintain tolerance to bacteria that populate the gut. We propose to elucidate the mechanisms of intestinal tolerance to bacterial components. Our findings will improve understanding of intestinal inflammatory diseases such as necrotizing enterocolitis, and suggest therapies aimed at augmenting tolerance to intestinal bacteria.